Clinical and Pathologic Features of Congenital Myasthenic Syndromes Caused by 35 Genes—A Comprehensive Review

Congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders characterized by impaired neuromuscular signal transmission due to germline pathogenic variants in genes expressed at the neuromuscular junction (NMJ). A total of 35 genes have been reported in CMS (AGRN, ALG14, ALG2, CHAT, CHD8, CHRNA1, CHRNB1, CHRND, CHRNE, CHRNG, COL13A1, COLQ, DOK7, DPAGT1, GFPT1, GMPPB, LAMA5, LAMB2, LRP4, MUSK, MYO9A, PLEC, PREPL, PURA, RAPSN, RPH3A, SCN4A, SLC18A3, SLC25A1, SLC5A7, SNAP25, SYT2, TOR1AIP1, UNC13A, VAMP1). The 35 genes can be classified into 14 groups according to the pathomechanical, clinical, and therapeutic features of CMS patients. Measurement of compound muscle action potentials elicited by repetitive nerve stimulation is required to diagnose CMS. Clinical and electrophysiological features are not sufficient to identify a defective molecule, and genetic studies are always required for accurate diagnosis. From a pharmacological point of view, cholinesterase inhibitors are effective in most groups of CMS, but are contraindicated in some groups of CMS. Similarly, ephedrine, salbutamol (albuterol), amifampridine are effective in most but not all groups of CMS. This review extensively covers pathomechanical and clinical features of CMS by citing 442 relevant articles.


Overview of Congenital Myasthenic Syndromes (CMS)
CMS are caused by defects in molecules expressed at the neuromuscular junction (NMJ) and are characterized by defective neuromuscular signal transduction [1][2][3]. As of January 2023, germline pathogenic variants in 35 genes have been reported (AGRN, ALG14, ALG2, CHAT, CHD8, CHRNA1, CHRNB1, CHRND, CHRNE, CHRNG, COL13A1, COLQ, DOK7, DPAGT1, GFPT1, GMPPB, LAMA5, LAMB2, LRP4, MUSK, MYO9A, PLEC, PREPL, PURA, RAPSN, RPH3A, SCN4A, SLC18A3, SLC25A1, SLC5A7, SNAP25, SYT2, TOR1AIP1, UNC13A, and VAMP1) (Figure 1). The causative genes can be classified into 14 groups depending on the pathomechanical features. Clinical features and therapeutic strategies are shared between some or all groups of CMS, but some features and therapies are unique to specific groups of CMS. Some therapies are ineffective or even contraindicated in some groups. Clinical features and therapeutic responses are generally difficult to predict a defective molecule. Clinical features of CMS are characterized by muscle fatigue, muscle weakness, muscle hypoplasia, and minor facial anomalies like low-set ears and high-arched palate in some patients. Autoimmune myasthenia gravis (MG) also compromises the NMJ signal transduction but is caused by autoantibodies against the acetylcholine receptor (AChR), muscle-specific receptor tyrosine kinase (MuSK), low-density lipoprotein receptor-related protein 4 (LRP4), or others. In contrast to MG, diurnal fluctuation of muscle strength and muscle fatigue are not always observed in CMS. In some CMS patients, day-to-day fluctuation of muscle strength is prominent. Diurnal fluctuation of external ophthalmoplegia is associated with diplopia in MG, but not always in CMS. This is likely due to the presence of external ophthalmoplegia since infancy, which enables compensation for the fluctuating visual axes. Some lack eye symptoms and are referred to as limb-girdle CMS. Most CMS patients develop the disease before age 2 years, but, in some patients, symptoms develop immediately after birth but temporarily subside thereafter until adolescence or adulthood. Including these neonatal transient patients, CMS can develop at any age including adolescence and adulthood. Some patients with CHAT-CMS, LAMB2-CMS, SLC5A7-CMS, SNAP25-CMS, UNC13A-CMS, DPAGT1-CMS, ALG2-CMS, MYO9A-CMS, SLC25A1-CMS, and PURA-CMS exhibit developmental delay. This can be caused by defective cholinergic neurotransmission in the central nervous system (CNS) or hypoxic brain injury due to repeated apneustic attacks, but the exact mechanisms remain to be elucidated. Episodic apnea is frequently reported in CHAT-CMS, COLQ-CMS, and SCN4A-CMS, but is also observed in other groups of CMS. Siblings of CMS patients with episodic apnea sometimes die with a diagnosis of sudden infantile Clinical features of CMS are characterized by muscle fatigue, muscle weakness, muscle hypoplasia, and minor facial anomalies like low-set ears and high-arched palate in some patients. Autoimmune myasthenia gravis (MG) also compromises the NMJ signal transduction but is caused by autoantibodies against the acetylcholine receptor (AChR), muscle-specific receptor tyrosine kinase (MuSK), low-density lipoprotein receptor-related protein 4 (LRP4), or others. In contrast to MG, diurnal fluctuation of muscle strength and muscle fatigue are not always observed in CMS. In some CMS patients, day-to-day fluctuation of muscle strength is prominent. Diurnal fluctuation of external ophthalmoplegia is associated with diplopia in MG, but not always in CMS. This is likely due to the presence of external ophthalmoplegia since infancy, which enables compensation for the fluctuating visual axes. Some lack eye symptoms and are referred to as limb-girdle CMS. Most CMS patients develop the disease before age 2 years, but, in some patients, symptoms develop immediately after birth but temporarily subside thereafter until adolescence or adulthood. Including these neonatal transient patients, CMS can develop at any age including adolescence and adulthood. Some patients with CHAT-CMS, LAMB2-CMS, SLC5A7-CMS, SNAP25-CMS, UNC13A-CMS, DPAGT1-CMS, ALG2-CMS, MYO9A-CMS, SLC25A1-CMS, and PURA-CMS exhibit developmental delay. This can be caused by defective cholinergic neurotransmission in the central nervous system (CNS) or hypoxic brain injury due to repeated apneustic attacks, but the exact mechanisms remain to be elucidated. Episodic apnea is frequently reported in CHAT-CMS, COLQ-CMS, and SCN4A-CMS, but is also observed in other groups of CMS. Siblings of CMS patients with episodic apnea sometimes die with a diagnosis of sudden infantile death syndrome (SIDS) [4,5]. Continuous monitoring of apnea is required for these patients.

Inheritance
Autosomal dominant inheritance or hemiallelic pathogenic variants are observed in SC-CMS, SNAP25-CMS [15,54], PURA-CMS [55], and some [9,56,57] but not the other [58][59][60] patients of SYT2-CMS. In contrast, other groups of CMS show autosomal recessive inheritance or require pathogenic loss-of-function variants in two alleles. SCCMS is caused by a gain of function missense variant in a single allele, because prolonged AChR channel openings in half of AChRs at the NMJ are sufficient to cause the SCCMS. Both SYT2-CMS and SNAP25-CMS exhibit LEMS-like CMS, and are likely to be caused by dominant negative effects. PURA-CMS is likely to be caused by a hemiallelic loss-of-function.
Ephedrine and salbutamol (albuterol) are effective in many groups of CMS including endplate AChR deficiency caused by pathogenic variants in a large number of genes, as well as in DOK7-CMS. Sympathetic nerve innervates the NMJ and facilitates the NMJ signal transmission, which is likely to be a pharmaceutical mechanism of the effects of ephedrine and salbutamol (albuterol) [72]. Ephedrine and salbutamol (albuterol) are also effective in some patients with SCCMS and COLQ-CMS, which is likely to compensate for AChR deficiency due to endplate myopathy.
Amifampridine blocks voltage-gated potassium channel at the nerve terminal to potentiate the action potential of the motor nerve, and enhances calcium entry into the nerve terminal, which subsequently facilitates release of ACh into the synaptic space. Amifampridine is effective for LEMS-like CMS, which is characterized by compromised SNARE complex. In addition, amifampridine is effective for many groups of CMS except for SCCMS, AGRN-CMS, SLC5A7-CMS, SLC25A1-CMS. Amifampridine is also effective in some patients with COLQ-CMS [62,73], although the pharmacological mechanisms remain unknown.
In 27 pregnancies in 16 CMS patients, all patients continued to take drugs. The symptoms were worsened in 63% of the pregnancies but were subsided after delivery [82].

Physiological Aspects of Neuromuscular Signal Transmission
It is essential to understand the physiology of signal transduction at the NMJ to recognize the pathomechanisms of CMS (Figure 1). The action potential of the spinal motor neuron is delivered to the nerve terminal, and activates the P/Q-type calcium channel (CACNA1A). The calcium ions bind to two C2B domains of synaptotagmin 2 (SYT2), and activate the SNARE complex to fuse synaptic vesicles containing ACh to the presynaptic membrane [83]. ACh is then released to the 70-nm synaptic space. ACh released from the nerve terminal is hydrolyzed by AChE in synaptic space, and two molecules of ACh that were not captured by AChE bind to AChR to open a cationic ion channel pore. ACh dissociated from AChR is hydrolyzed to choline by AChE. The generated choline in the synaptic space is up taken by high affinity choline transporter (ChT, SLC5A7) expressed in the membrane of the nerve terminal [84]. Choline acetyltransferase (ChAT, CHAT) in the nerve terminal generates ACh from up taken choline and acetyl-CoA. Vacuolar proton ATPase embedded in the synaptic vesicle generates a proton gradient, which drives an import of ACh into the synaptic vesicle through vesicular acetylcholine transporter (vAChT, SLC18A3) [85].
Adult-type AChR is comprised of the α1 (CHRNA1), β1 (CHRNB1), δ (CHRND), and ε (CHRNE) subunits ( Figure 2). Two α1 subunits and each of β1, δ, and ε subunits make a pentameric AChR (α1 2 β1δε). Embryonic AChR of a α1 2 β1δγ pentamer will be addressed in a section for endplate AChR deficiency. AChR subunits have four transmembrane domains (M1, M2, M3, and M4), and their N-and C-terminals are on the extracellular side. The second transmembrane domain, M2, makes an ion channel pore. The N-terminal regions of AChR subunits make a large extracellular complex, and ACh binds to the interfaces between α1-ε subunits and α1-δ subunits. AChR is a cation-nonselective ion channel that can pass through all cationic ions of Na + , Ca 2+ , and Mg 2+ . As Na + is the major cation in the extracellular space, Na + is the major source to make an endplate potential (EPP). The conductance and the burst duration of fetal AChR are~70% and~240% of those of adult-type AChR at the human endplate [86].
presynaptic membrane [83]. ACh is then released to the 70-nm synaptic space. ACh released from the nerve terminal is hydrolyzed by AChE in synaptic space, and two molecules of ACh that were not captured by AChE bind to AChR to open a cationic ion channel pore. ACh dissociated from AChR is hydrolyzed to choline by AChE. The generated choline in the synaptic space is up taken by high affinity choline transporter (ChT, SLC5A7) expressed in the membrane of the nerve terminal [84]. Choline acetyltransferase (ChAT, CHAT) in the nerve terminal generates ACh from up taken choline and acetyl-CoA. Vacuolar proton ATPase embedded in the synaptic vesicle generates a proton gradient, which drives an import of ACh into the synaptic vesicle through vesicular acetylcholine transporter (vAChT, SLC18A3) [85].
Adult-type AChR is comprised of the α1 (CHRNA1), β1 (CHRNB1), δ (CHRND), and ε (CHRNE) subunits ( Figure 2). Two α1 subunits and each of β1, δ, and ε subunits make a pentameric AChR (α12β1δε). Embryonic AChR of a α12β1δγ pentamer will be addressed in a section for endplate AChR deficiency. AChR subunits have four transmembrane domains (M1, M2, M3, and M4), and their N-and C-terminals are on the extracellular side. The second transmembrane domain, M2, makes an ion channel pore. The N-terminal regions of AChR subunits make a large extracellular complex, and ACh binds to the interfaces between α1-ε subunits and α1-δ subunits. AChR is a cation-nonselective ion channel that can pass through all cationic ions of Na + , Ca 2+ , and Mg 2+ . As Na + is the major cation in the extracellular space, Na + is the major source to make an endplate potential (EPP). The conductance and the burst duration of fetal AChR are ~70% and ~240% of those of adult-type AChR at the human endplate [86]. Depolarization by EPP elicits the opening of skeletal muscle voltage-gated sodium channel (NaV1.4, SCN4A) to generate a muscle action potential. NaV1.4 is expressed throughout the muscle fiber but is enriched at the motor endplate. Muscle action potential goes into the T tubules, where depolarization is sensed by L-type calcium channel (CaV1.1, CACNA1S), which constitutes dihydropyridine receptor (DHPR) with other molecules. Depolarization by EPP elicits the opening of skeletal muscle voltage-gated sodium channel (Na V 1.4, SCN4A) to generate a muscle action potential. Na V 1.4 is expressed throughout the muscle fiber but is enriched at the motor endplate. Muscle action potential goes into the T tubules, where depolarization is sensed by L-type calcium channel (Ca V 1.1, CACNA1S), which constitutes dihydropyridine receptor (DHPR) with other molecules. DHPR is coupled to ryanodine receptor (RyR, RYR1), and RyR releases Ca 2+ from the sarcoplasmic reticulum (SR). Sarcoplasmic Ca 2+ binds to troponin and displaces tropomyosin that covers the binding sites of actin for the myosin head to contract the muscle fibers.

Thirty-Five Genes in 14 Groups of CMS
CMS are caused by 35 genes, which can be grouped into 14 groups, based on pathomechanisms. Pathomechanisms, clinical features, and therapies are widely variable from category to category. Embryonic AChR is composed of α1 2 β1δγ. The embryonic γ subunit is substituted by the adult-type ε subunit after birth to make α1 2 β1δε. The adult-type α1 2 β1δε has a higher conductance and a shorter opening time compared to the embryonic α1 2 β1δγ [86]. Biallelic lack of the ε subunit or (CHRNE) in CMS patients can be compensated for by the γ subunit and is not fatal [16,[103][104][105]. The expression of γ-AChR is also observed when the expression of ε-AChR is markedly reduced. In contrast, biallelic lack of the α1, β1, and δ subunits (CHRNA1, CHRNB1, and CHRND, respectively) cannot be compensated for by another subunit, and is fatal. Hemiallelic null variants in CHRNA1, CHRNB1, and CHRND are asymptomatic if the other allele has no pathogenic variant, whereas biallelic null variants are observed only in CHRNE. Missense variants in genes encoding the α1, β1, δ, and ε subunits (CHRNA1, CHRNB1, CHRND, and CHRNE, respectively) that markedly reduce the cell surface expression of AChR cause endplate AChR deficiency [106,107]. Some missense variants in these genes simultaneously cause endplate AChR deficiency, as well as slow or fast channel myasthenic syndrome (SCCMS or FCCMS). Especially, in FCCMS, the reduction in channel opening events to~50% alone is not pathogenic, but becomes pathogenic when the expression level is also reduced to~50% [108].
Aberrant splicing of CHRNA1 causes an unusual form of endplate AChR deficiency. CHRNA1 has a 75-nt exon P3A between exons 3 and 4 that is unique to human and anthropoids. An exon P3A-skipped CHRNA1 transcript makes normal AChR, whereas an exon P3A-included CHRNA1 transcript cannot form AChR. In human skeletal muscle, P3A(+) and P3A(-) transcripts are generated at a ratio of 1:1, although the physiological significance remains unknown. Pathogenic variants in exon P3A and its preceding intron exclusively include exon P3A in pre-mRNA splicing, and the generated P3A(+) transcript causes endplate AChR deficiency [109][110][111].
Pathogenic variants in RAPSN encoding rapsyn also cause endplate AChR deficiency. Some missense variants in RAPSN retain self-clustering of rapsyn [112], whereas the others do not [113]. Rapsyn phosphorylated by the agrin-LRP4-MuSK pathway forms submembranous network by self-clustering and also activates the E3 ligase activity, which is compromised by a founder variant, p.N88K in RAPSN [114]. Four siblings born from consanguineous parents carried pathogenic homovariants in RAPSN (c.491G>A, p.R146H) [115]. However, only two of them were affected by CMS, whereas the other two were not. The two affected CMS siblings additionally had homovariants in AK9. AK9 encodes one of nine adenylate kinases, and catalyzes a conversion between nucleotide diphosphate and nucleotide triphosphate. The identified variant in AK9 was a single nucleotide variation (SNV) at 14 nucleotides upstream to the boundary of intron 5 and exon 6. This variant may make a de novo translational start site, but no experimental evidence was provided. In addition, as the same RAPSN variant was reported in another CMS patient [116], lack of phenotypes in the two siblings without AK9 remains unknown.

Clinical Features and Therapies
Endplate AChR deficiency caused by pathogenic variants in CHRNA1, CHRNB1, CHRND, and CHRNE have been repeatedly reported since 1996 [117]. Frameshifting and nonsense variants are recognized to be pathogenic even without expression studies, but pathogenic missense variants in CHRNA1, CHRNB1, CHRND, or CHRNE may cause (i) reduced AChR expression, (ii) SCCMS, or (iii) FCCMS. Except for a dominantly inherited hemiallelic missense variant in a pedigree, which causes SCCMS, the effects of missense variants in CHRNE cannot be differentiated without expression studies. Lack of expression studies in most pathogenic variants prevents us from counting the number of patients or original articles with endplate AChR deficiency. However, endplate AChR deficiency and FCCMS have essentially the same clinical features, which are also similar to myasthenia gravis. In contrast to myasthenia gravis, endplate AChR deficiency is present in embryogenesis in patients with pathogenic variants in CHRNA1, CHRNB1, and CHRND, or is present from birth in patients with pathogenic variants in CHRNE. These are likely to account for minor facial anomalies, muscle hypoplasia, and lack of diplopia.
ChEIs are generally effective for endplate AChR deficiency irrespective of defective genes. We, however, should be aware that excessive administration of ChEIs causes an iatrogenic pathology similar to endplate AChE deficiency due to pathogenic variants in COLQ stated below. Ephedrine and salbutamol (albuterol) are also generally effective for endplate AChR deficiency [144]. The effects of adrenergic agonists are likely due to the innervation of sympathetic nerve to the NMJ and the facilitation of the NMJ signal transmission by the sympathetic nerve [72]. In addition, amifampridine is also effective for endplate AChR deficiency [144,145]. Loss-of-function variants of CHRNG cause EVMSP (Escobar syndrome) and LMPS, both of which are characterized by arthrogryposis multiplex congenita (AMC) and pterygium likely due to the embryonic immobility [35][36][37]. Escobar syndrome takes a benign non-progressive course. A case of Escobar syndrome with uniparental disomy, in which a specific region of both alleles arises from a single parent, is reported [146]. FADS and LMPS are spectrum disorders [147]. Pathogenic variants of CHRNA1 [148], CHRND [148], RAPSN [39,40,147,148], DOK7 [147,149], and SLC18A3 [150] also cause LMPS/FADS. The phenotypes are again likely to be caused by embryonic immobility due to defective NMJ signal transmission.

Clinical Features and Therapies
Escobar syndrome has been reported in 101   SCCMS is caused by pathogenic missense variants in one allele of CHRNA1, CHRNB1, CHRND, and CHRNE, encoding the AChR α1, β1, δ, and ε subunits, respectively, and shows autosomal dominant inheritance. A case of autosomal recessive SCCMS was also reported [162,163]. Pathogenic missense variants of SCCMS can be classified into two categories. The first category includes pathogenic missense variants at the extracellular domain especially at the ACh-binding site and at the first transmembrane domain, M1. These variants delay the dissociation of ACh from AChR. The second category includes pathogenic missense variants at the second transmembrane domain, M2, that forms the ion channel pore [164,165]. Three mechanisms may result in defective NMJ signal transmission in SCCMS. First, prolonged openings of AChR ion channel increase the intracellular Na + concentration and depolarize the resting membrane potential, which reduces the amplitude of an endplate potential (EPP) and makes the muscle sodium channel (Na V 1.4) difficult to sense an EPP. Second, as AChR is a cation-non-selective ion channel, prolonged openings of AChR allow excessive influx of Ca 2+ ions, that cause endplate myopathy [166]. Ca 2+ ions constitute 7% of the endplate current of adult-type εAChR, which is higher than fetal γ-AChR. In two pathogenic variants in SCCMS (CHRNE p.T284P [164] and CHRNE p.V279F [27]), the permeability of Ca 2+ ions was increased 1.5-to 2.0-folds, which were likely to accelerate endplate myopathy [167]. Third, prolonged openings of AChR desensitize AChR [168]. AChR is physiologically desensitized by prolonged existence of ACh. Desensitized AChR does not respond to ACh and cannot generate EPP anymore. The structure of desensitized Torpedo AChR was recently solved [169]. In the desensitized state, the two agonist-binding sites between the α-δ and α-ε subunits of AChR are rotated counterclockwise perpendicular to the membrane, and the structure of the extracellular end of the M4 helix of the α subunit that interfaces with the δ subunit becomes much different.
FCCMS is kinetically opposite to SCCMS. Pathogenic missense variants can be classified into three categories. The first category includes pathogenic missense variants at the extracellular domain including the ACh-binding site of AChR [170]. Interestingly, detailed kinetic analyses reveal that most of the variants at the ACh-binding site [107,[170][171][172] affect the ion channel gating rather than ACh-binding to AChR. However, the other variants affect ACh-binding alone [171] or both ACh-binding and the ion channel gating [173]. The second category is comprised of pathogenic variants in the long cytoplasmic loop between the second and third transmembrane domains (M3 and M4). These variants destabilize the open channel state [86,174,175]. The third category is a pathogenic missense variant at the third transmembrane domain (M3) [176]. The enlarged amino acid in the M3 domain displaces the second transmembrane domain (M2) and narrows the ion channel pore made by the M2 domains of five subunits.

Clinical Features and Therapies
SCCMS has been reported in 34 original articles since 1995 [27,31,162,163,165,168,. As observed in other autosomal dominant disorders, the onset of SCCMS can be in adolescence or adulthood. Adult-onset patients tend to have mild phenotypes. Weakness of the extensor muscles of the upper limbs is frequently observed in SCCMS, although the underlying mechanisms remain unknown. Weakness of the extensor muscles of the upper limbs is also reported in 10 out of 15 patients with DOK7-CMS [66]. R-CMAP in response to a single nerve stimulus is observed in SCCMS, as in COLQ-CMS and PURA-CMS. A review of 60 SCCMS patients showed that R-CMAP was observed when the opening burst durations of mutant AChR were increased 8.68-fold on average compared to those of wild-type AChR, whereas R-CMAP was not observed when they were increased 3.84-fold on average [198]. Based on knowledge that sodium channel blockers also block AChR ion channel to some extent, shortening of abnormally prolonged AChR channel openings by an antiarrhythmic, quinidine [74] and an selective serotonin reuptake inhibitor (SSRI), fluoxetine [76] was reported by a single channel recordings of SCCMS-AChRs. Indeed, both quinidine [75] and fluoxetine [76] are effective for SCCMS. Amelioration of endplate myopathies in SC-CMS requires several months, and immediate effects of quinidine and fluoxetine are not usually observed. A review of 15 SCCMS patients showed that most patients improved by quinidine or fluoxetine, but the effects were not observed for respiratory insufficiency and palpebral ptosis [193]. In their report, 2 out of 6 SCCMS patients with quinidine developed adverse reactions of hypersensitivity reaction and impaired liver function. In 10 SCCMS patients treated with fluoxetine, 7 patients showed clear response, whereas 3 patients either showed adverse effects of serotonergic crisis, lethargy, and hypotension, or could not tolerate higher dose (40 mg/day) [193]. Similarly, in the initial report of fluoxetine for SCCMS, one of two patients had insomnia, drowsiness, and anorexia [76]. A SCCMS patient suffered suicidal ideation soon after commencing fluoxetine, which is well-recognized concern with fluoxetine [205]. Another review of 60 SCCMS patients showed that patients showing good responses to quinidine or fluoxetine started treatment at 11.6 years after the onset of symptoms on average, whereas patients without good responses started treatment at 30.7 years after the onset on average [198]. Although both quinidine and fluoxetine minimally shorten the openings of wild-type AChR, worsening of symptoms was reported in a patient with RAPSN-CMS by fluoxetine that was prescribed for depression [77]. ChEIs and amifampridine are ineffective in most SCCMS patients [31,184,188,193], but ChEIs improved the symptoms of a patient with SCCMS [185]. ChEIs presumably enhance the desensitization of AChRs and reduce the number of available AChRs that can respond to ACh. In addition, the effects of ephedrine and salbutamol (albuterol) were reported in SCCMS [193,202,203], as well as in mouse models [206,207].
FCCMS has been reported in 13 original articles since 1996 [64,130,170,170,173,175,[208][209][210][211][212][213][214][215]. Biallelic pathogenic missense variants including small indels either cause AChR deficiency or FCCMS. To differentiate the two types of pathologies, microelectrode studies and/or single channel recordings of the biopsied skeletal muscle, and/or single channel recordings of mutant AChR expressed on cultured cells, are required. The unavailability of these techniques is likely to account for the rarity of FCCMS. Indeed, FCCMS has been reported from only three laboratories in the world. Pathogenic variants of FCCMS have been reported in CHRNA1, CHRND, and CHRNE, but not in CHRNB1. Although only the β subunit does not contribute to make a ACh-binding site, missense variants in CHRNB1 can possibly cause FCCMS. Although the differentiation of FCCMS and AChR deficiency is challenging, similar therapies can be applied to both diseases. FCCMS patients respond to ChEIs [2,130,173,200,213], amifampridine [2,213], and salbutamol (albuterol) [216]. Ephedrine is also likely to be effective for FCCMS, but its effect has not been reported. Favorable responses to ChEIs and amifampridine may not necessitate the use of other drugs.

Synaptic CMS (COLQ, LAMB2, and COL13A1) 4.4.1. Pathomechanisms
One, two, and four molecules of AChE enzyme make globular forms of AChE that are named G 1 , G 2 , and G 4 , respectively. In addition, triple helical collagen Q (ColQ) binds to 4, 8, and 12 molecules of AChE and makes asymmetric forms of AChE named A 4 , A 8 , and A 12 , respectively. Asymmetric forms of AChE are enriched at the NMJ. ColQ has three domains. First, the proline-rich attachment domain (PRAD) at the N terminal end is enriched in prolines. The tetrameric forms of AChE bind to PRAD, and three ColQ strands make A 12 -AChE. Second, the collagen domain in the middle of ColQ has prolines at every three residues like other collagens, and makes a stable triple helical structure. The collagen domain has two regions enriched in positively charged basic amino acids, where heparan sulfate proteoglycans (HSP) including perlecan bind [217]. The two regions are named HSP-binding domain (HSPBD) [218]. Third, the C-terminal domain (CTD) of ColQ is enriched in charged amino acids and cysteines, and makes a globular form. CTD of ColQ binds to MuSK [102,219,220]. Asymmetric forms of AChE are generated in the Golgi apparatus, excreted to the synaptic space, and are anchored to the synaptic basal lamina by binding of ColQ to HSP and MuSK.
Loss-of-function variants of COLQ cause endplate AChE deficiency [221][222][223][224][225]. Although the roles of ColQ at the NMJ have been well analyzed, ColQ is also expressed in other tissues including brain, testis, and heart. The roles of ColQ in other tissues, however, remain unknown, and Colq-deficient mice show no phenotypes other than endplate AChE deficiency [226,227]. In contrast to COLQ, no pathogenic variants have been reported in ACHE in any diseases. AChE plays essential roles in the cholinergic synapses in the CNS. Loss-of-function of AChE is thus likely to be fatal in humans. Although there is no relevance to human diseases, p.H322N (rs1799805) in ACHE determines the YT blood group [228]. Pathogenic variants of COLQ are classified into three categories [224]. First, pathogenic variants in PRAD impair the binding of AChE to PRAD. Second, pathogenic variants in the collagen domain impair the formation of the triple helix. Most of them are nonsense or frameshifting variants. Third, pathogenic variants at CTD impair anchoring of ColQ to the NMJ by inhibiting the binding of ColQ to MuSK [225,229].
Although both endplate AChE deficiency and SCCMS are caused by excessive openings of AChR, the mechanisms of defective NMJ signal transmission are not identical. Two mechanisms are similar between the two diseases. First, depolarization of the resting membrane potential reduces the amplitude of EPP, and small EPP cannot activate the skeletal muscle sodium channel. Second, AChRs are desensitized by the prolonged presence of ACh in endplate AChE deficiency and prolonged openings of AChRs in SCCMS. In contrast to SCCMS, however, endplate myopathy due to excessive influx of Ca 2+ ions are not observed in endplate AChE deficiency, because the nerve terminal becomes small, and the terminal Schwann cells invaginate into the synaptic space, both of which reduce the number of releasable ACh quanta.
Laminins-221, -421, and -521, all of which include β2-laminin (LAMB2), are expressed at the NMJ. Lamins are heterotrimeric extracellular matrix molecules made of α, β, and γ subunits, and are key molecules constituting the synaptic basal lamina [230]. Laminins play a critical role in maintenance of the NMJ, and organization of synaptic vesicle release sites known as active zones. Laminins-221, -421, and -521 are made of α2-, α4-, and α5-laminins, respectively, as well as of β2-, and γ1-laminins. Laminins at the NMJ play essential roles in the juxtaposition of presynaptic and postsynaptic structures and the placement of the terminal Schwann cells at the NMJ. β2-Laminin directly binds to P/Q-and N-type voltage-gated calcium channel (VGCC) [231][232][233], and is essential for the formation and organization of presynaptic active zones [234]. β2-Laminin is also expressed in renal glomeruli and eyes. Pathogenic variants in LAMB2 cause Pierson syndrome [235] and nephrotic syndrome type 5 [236]. Pierson syndrome is characterized by congenital nephrotic syndrome and a complex maldevelopment of the eye with lens abnormalities, atrophy of the ciliary muscle, corneal changes, and retinal changes. Pathological variants of LAMB2 were reported in a CMS patient with Pierson syndrome [65]. Ultrastructural analysis of the biopsied muscle showed marked reduction in the size of the nerve terminals, invagination of the synaptic space by the processes of Schwann cells, and moderate simplification of postsynaptic folds. Electrophysiological examinations showed marked reduction in quantal release of ACh from the nerve terminal. Lamb2-deficient mice also show similar phenotypes at the NMJ [237].
Collagen 13α1 (COL13A1) enriched at the NMJ has a single transmembrane domain and plays an essential role in the maturation and maintenance of AChR at the NMJ [238]. A frameshifting variant of COL13A1 causes CMS [239]. Introduction of the pathogenic variants into C2C12 myotubes reduced AChR clustering [239]. Col13a1-deficient mice show abnormal formation of the NMJ [238,240], as well as craniofacial malformations and a reduction in cortical bone mass in aged mice [241].

Clinical Features and Therapies
COLQ-CMS has been reported in 30 original articles since 1998 [62,73,78,140,141,221,222,225,. Interestingly, a grandmother and a father of two siblings with COLQ-CMS carried a heterozygous truncation variant of COLQ, and showed congenital ptosis [246]. Although the presence of a pathogenic variant on another allele could not be excluded, a heterozygous variant of COLQ might have caused ptosis. Initial symptoms of COLQ-CMS are mostly ophthalmoplegia, respiratory insufficiency, and weak crying at birth. Follow-up of 15 patients with COLQ-CMS aged 3 to 48 years for up to 10 years showed that 80% of patients were still ambulant and 87% had no respiratory difficulties [253]. A report of 22 patients with COLQ-CMS indicated proximal dominant muscle weakness that was characteristic of limb-girdle-type myasthenia as in DOK7-CMS [247]. Fluctuating scoliosis due to truncal muscle weakness is later changed to severe scoliosis, which is uniquely observed in COLQ-CMS and DOK7-CMS [249]. Palpebral ptosis and external ophthalmoplegia are observed in about half of COLQ-CMS patients [247,253]. Diurnal fluctuation and progression of muscle weakness are observed in about half of the patients [253]. Delayed pupillary response is characteristic of COLQ-CMS but is observed in only 25% of the patients [247]. Repetitive CMAP, which is also observed in SCCMS and PURA-CMS, is observed in about half of the patients [253]. In COLQ-CMS, globular forms of AChE and butyrylcholinesterase at the motor endplate catalyze ACh, and blocking of these enzymes by ChEIs can sometimes cause respiratory arrest [62][63][64]. ChEIs had no long-term effects in 22 patients with COLQ-CMS but showed short-time effects in 4 patients [247]. Ephedrine and salbutamol (albuterol) are effective for COLQ-CMS [264][265][266]. Especially, in two patients, ephedrine showed marked effects [264], although the underlying mechanisms remain unknown. The effects of amifampridine are also reported [62,73], but the mechanisms again remain elusive. The effect of fluoxetine that is used for SCCMS was also reported in a patient with COLQ-CMS [78]. Although fluoxetine minimally shortens the channel opening time of wild-type AChR [74], a slight reduction in AChR openings was likely to be sufficient for the patient.
LAMB2-CMS was reported in a 20-year-old female with Pierson syndrome in 2009 [65]. The patient had repeated respiratory distress since birth, miosis, and severe proteinuria. Development of motor functions were delayed, but proteinuria was improved by rental transplantation at age 7 years. Palpebral ptosis, external ophthalmoplegia, and proximal muscle weakness were noted. RNS reduced CMAP by 24%. ChEI worsened her muscle weakness, and respiratory support was required. Ephedrine was effective.

Sodium Channel CMS (SCN4A) 4.5.1. Pathomechanisms
Loss-of-function variants of SCN4A cause CMS [79,81,270], whereas gain-of-function variants of SCN4A cause hyperkalemic periodic paralysis [271], hypokalemic periodic paralysis [271], potassium-aggravated myotonia congenita [272], and paramyotonia congenita [273]. Loss-of-function variants of SCN4A in CMS shift the fast inactive curve toward hyperpolarized states and make Na V 1.4 inactive even at the resting membrane potential. Na V 1.4 ion channel opens in response to the first depolarization stimulus, but not to the second or later depolarization stimuli because of accelerated transition to a fast inactive state. This also causes decremental CMAP response to RNS. In contrast to SCN4A-CMS, gain-of-function variants in hyperkalemic periodic paralysis, hypokalemic periodic paralysis, potassium-aggravated myotonia congenita, and paramyotonia congenita shift the fast inactivation curve toward depolarized states. This allows repeated openings of Na V 1.4 or allows leakage of Na + even in a closed state. In SCN4A-CMS and in some CMS patients with defective recycling of ACh (CHAT-CMS, SLC18A3-CMS, SLC5A7-CMS, and PREPL-CMS), a high-frequency nerve stimulation is required to elicit a decremental CMAP response, and episodic muscle weakness is observed.

Clinical Features and Therapies
SCN4A-CMS has been reported in 6 patients since 2003 [79][80][81]140,270,274]. SCN4A-CMS shows frequent episodes of respiratory arrest, bulbar paralysis, and muscle weakness that persist 30 to 60 min. In the intermittent phase, mild facial, truncal, and limb muscle weakness, as well as external ophthalmoplegia, are observed. Analysis of 278 patients with sudden infantile death syndrome (SIDS) revealed 4 patients with SCN4A-CMS [5]. ChEIs are either effective [79,142,274] or ineffective [270]. In addition, a SCN4A-CMS patient showed marked cholinergic adverse effects with a small amount of ChEI [80]. Salbutamol (albuterol) was effective in a single patient [142]. Similarly, acetazolamide was either effective [79,80] or ineffective [81] to prevent episodic muscle weakness. Agrin (AGRN) is a large molecule secreted from the nerve terminal with a molecular weight of~200 kDa, and carries binding domains for laminins, neural cell adhesion molecule (NCAM), α-dystroglycan, and LRP4. Functionally characterized pathogenic variants of AGRN invariably impair AChR clustering. However, three pathologies exist depending on the affected domains: (i) impairment of MuSK phosphorylation, (ii) accelerated degradation of agrin, (iii) impaired anchoring of agrin to the NMJ [275].
The third β propeller domain of LRP4 binds to agrin. Pathogenic variants in this domain in CMS impair binding of LRP4 to agrin and MuSK, reduce MuSK phosphorylation, and compromise AChR clustering [71]. Pathogenic variants in this domain are also reported in sclerosteosis type 2 (SOST2), which is characterized by cortical hyperostosis [276]. CMS variants affect agrin-LRP4-MuSK signaling but not Wnt signaling, whereas SOST2 variants affect Wnt signaling but not agrin-LRP4-MuSK signaling. Analysis of additional artificial variants revealed that variants at the periphery of the third β propeller domain exclusively affect agrin-LRP4-MuSK signaling, whereas variants at the center of the domain exclusively affect Wnt signaling [71]. Pathogenic variants of the other domains of LRP4 are also reported in another bone disorder, Cenani-Lenz syndactyly syndrome [277]. Thus, pathogenic variants of LRP4 either affect agrin-LRP4-MuSK signaling or Wnt signaling.
Pathogenic variants of MUSK either reduces cell membrane expression of MuSK without affecting agrin-mediated phosphorylation of MuSK [278], or markedly reduces MuSK phosphorylation and AChR clustering [279].

Clinical Features and Therapies
AGRN-CMS has been reported in 13 original articles since 2009 [6,141,142,274,275,[285][286][287][288][289][290][291][292]. Two patients with AGRN-CMS reported in 2009 were 42-year-old female and 36-year-old male in a single pedigree, who had mild limb muscle weakness and unilateral ptosis since childhood [285]. ChEIs and amifampridine were ineffective. Most AGRN-CMS patients similarly develop muscle weakness since childhood, and the symptoms range from mild muscle weakness in lower limbs to severe muscle weakness that requires respiratory support. Again, ChEIs and amifampridine are ineffective or mildly effective. On the other hand, salbutamol (albuterol) was effective in 10 out of 12 AGRN-CMS patients [287]. Similarly, ephedrine was effective in a single patient [288]. Biallelic null variants of AGRN caused FADS and gave rise to stillbirth at 30 weeks of gestation [293]. In addition, analysis of 262 patients with autism spectrum disorder (ASD) revealed hemiallelic null variants of AGRN [294]. However, as null variants of AGRN are observed in asymptomatic parents of AGRN-CMS patients, other genetic or environmental factors are likely to be required to be associated with ASD. In addition, pathogenic AGRN variants were identified in hereditary motor neuropathy, in whom jitters by SFEMG were increased [295].
LPR4-CMS with compound heterozygous variants was reported in a single patient in 2014 [71]. The patient had respiratory distress at birth, and was dependent on a respirator up to age 6 years. Evaluations at ages 9 and 14 years showed mild external ophthalmoplegia and severe muscle weakness. ChEIs worsened muscle weakness.
DOK7-CMS has been reported in 34 [308][309][310][311][312][313][314][315][316][317][318][319][320][321][322][323][324]. A review of 15 patients with DOK7-CMS showed that the ages of onset were mostly from birth to infancy, and the oldest age of onset was 13 years [66]. All patients showed proximal and truncal muscle weakness, and scoliosis was frequently observed. In addition, distal muscle weakness, especially finger extensors, was observed in 12 patients. Similar, weakness of finger extensors is also observed in SCCMS. Muscle hypoplasia was present in about half of the patients; palpebral ptosis and external ophthalmoplegia in 11 patients; and facial and bulbar muscle weakness in 8 to 9 patients. DOK7-CMS is recognized as limb-girdle CMS, but ocular, facial, and bulbar weakness is frequently observed. The diagnosis of myasthenia gravis was erroneously given to 4 out of 15 patients, and others were diagnosed as congenital myopathy, metabolic myopathy, or mitochondrial myopathy. Although the causal relation remains unknown, siblings of DOK7-CMS had mitral valve insufficiency [322]. A total of six CMS patients were heterozygous for a truncation variant of DOK7 without any pathogenic variants on another allele [281,325]. Although the presence of a pathogenic variant on another allele could not be excluded, a heterozygous variant of DOK7 might cause CMS when unidentified genetic and/or environmental factor(s) coexisted. More such heterozygous patients may exist, but may be underestimated due to possible publication bias. The effects of ephedrine and salbutamol (albuterol) for DOK7-CMS have been repeatedly reported [64,[66][67][68][69]308,310,[315][316][317]321,324]. In addition the effect of a patch of tulobuterol, a β2 agonist, was reported in a case with DOK7-CMS [326]. On the other hand, ChEIs are ineffective or worsen muscle weakness [64,[66][67][68][69]. The effect of amifampridine was also reported [319]. Fluoxetine was effective in a patient with DOK7-CMS, who was misdiagnosed as SCCMS [327]. Administration of anti-DOK7 antibody that stimulated DOK7 was effective for a mouse model of DOK7-CMS carrying a pathogenic variant of the patient [328]. Although the mechanisms are unknown, a DOK7-CMS patient was responsive to steroid for 40 to 50 years [329]. In addition, 4 patients with FADS due to pathogenic variants of DOK7 were reported [147,149].

Pathomechanisms
Plectin is a 500-kD intermediate filament-binding protein that provides mechanical strength by acting as a crosslinking element of the cytoskeleton. In the skeletal muscle, plectin is expressed in sarcolemma and Z band. In the skin, plectin makes hemidesmosome. Pathogenic variants of PLEC cause epidermolysis bullosa simplex (EBS) [330] and autosomal recessive limb-girdle muscular dystrophy 17 (LGMD17) [331]. In patients with EBS and LGMD17, endplate AChR deficiency was reported [332][333][334]. A homozygous 9-bp deletion of the translational start site of PLEC caused LGMD17 in 3 patients in 3 pedigrees in Turkey without any evaluation of the NMJ [331]. The same variant, however, caused LGMD17 and endplate AChR deficiency in 4 patients in 4 pedigrees in Turkey [53], indicating that myasthenic symptoms might be masked by muscular dystrophy. Plectin is highly expressed at the NMJ, connects desmin and dystrophin-dystroglycan complex, binds to rapsyn-AChR complex, and stabilizes the NMJ structure [335]. Indeed, ultrastructural analyses show destruction and remodeling of the endplate [332].

Clinical Features and Therapies
PLEC-CMS has been reported in 22 [338]. A review of 117 PLEC-EBS patients carrying pathogenic variants in PELC showed that 14 patients also had CMS [339]. However, the authors observed the presence of CMS in 7 out of 15 patients in their own cohort of PLEC-EBS [339], indicating that CMS was likely to be underdiagnosed and that the prevalence of CMS was higher than reported. The prevalence of muscular dystrophy in PLEC-EBS was also high. The onsets of PLEC-CMS range from early childhood to age 26 years, and patients show limb muscle weakness, swallowing difficulty, respiratory insufficiency, palpebral ptosis, external ophthalmoplegia [53, 332,334,338]. As in most other patients with CMS, low-frequency RNS elicit decremental CMAP responses. ChEIs were ineffective in 3 patients [334], and effective in 3 other patients [338]. A combination of ChEIs and salbutamol (albuterol) was effective in 4 patients [53]. Amifampridine was effective in a case [338], and was ineffective in 2 other patients [334,338]. In addition, a case of PLEC-CHRNE-CMS who had both biallelic insertion of 36 bp in PLEC and biallelic frameshift in CHRNE showed EBS and CMS [333]. ChEIs and ephedrine were mildly effective for this case. Choline acetyltransferase (ChAT, CHAT) synthesizes ACh from choline and acetyl-CoA at the nerve terminal. Vesicular acetylcholine transporter (vAChT, SLC18A3) transports synthesized ACh to the synaptic vesicle. SLC18A3 is encoded within the first intron of CHAT. This nested gene structure is conserved from C. elegans. Loss-of-function variants of CHAT cause CMS with episodic apnea [340,341]. ChAT is also expressed at the cholinergic synapses in the CNS, and developmental delay that is observed in about half of CHAT-CMS patients can be accounted for either by defects in the cholinergic synapse in the CNS or by hypoxia due to episodic apnea. Parents of CHAT-CMS who carry a null variant in a single allele are asymptomatic, whereas no CHAT-CMS patients carry biallelic null variants. Thus, the reduction in the enzymatic activity of ChAT to 30-50% is predicted to cause CMS, whereas ChAT activities lower than 30% are lethal and more than 50% are asymptomatic [340,341].
Loss-of-function variants of SLC18A3 also cause CMS. Although the disease mechanisms have not been dissected in detail, failure to pack resynthesized ACh into synaptic vesicles is likely to be the cause of CMS.
A hemiallelic large scale DNA rearrangement at 10q11.2 including CHAT and SLC18A3 was observed in 41 patients with autism, developmental delay and/or intellectual disability, and multiple congenital malformations [342]. Muscle hypotonus, palpebral ptosis, and sleep apnea in these patients may be caused by haploinsufficiency of CHAT and SLC18A3. However, as stated above, as hemiallelic null variants are symptomatic in parents of CHAT-CMS, either complete lack of vAChT (SLC18A3) that is intact in CHAT-CMS or an unidentified pathogenic variant on the other allele may cause the disease. Indeed, in two patients with CMS, a hemiallelic large scale deletion at 10q11.2 region was unmasked by a pathogenic splicing variant in CHAT, or a pathogenic missense variant in SLC18A [343].
High affinity choline transporter (ChT, SLC5A7) expressed at the nerve terminal membrane uptakes choline to the nerve terminal. ChT is a homo-oligomeric membrane transporter. A hemiallelic frameshifting variant of SLC5A7 cause autosomal dominant distal hereditary motor neuropathy type VIIA (DHMN7A) [344,345]. DHMN7A is characterized by teen-age onset progressive distal limb muscle weakness and amyotrophy with vocal paralysis. Later, recessive pathogenic variants of SLC5A7 were reported to cause CMS [41]. Expression studies of cultured cells showed that dominantly inherited variants of SLC5A7 have dominant-negative effects [344], whereas recessively inherited variants have lossof-function effects [41,346], on choline uptake by ChT. Dominantly inherited variants of SLC5A7 are likely to inhibit the formation of homo-oligomers of ChT, whereas recessively inherited variants do not. However, it remains unknown why similar reductions of the ChT activity give rise to two different phenotypes of DHMN7A and CMS. Mice deficient for Slc5a7 dies in a few minutes after birth probably due to respiratory failure [347], and spinal motor neuron-specific rescue of Slc5a7 prolonged the knockout move and breath for~24 h after birth [348]. Hemizygous knockout of Slc5a7 in mice decreased cardiac ACh and showed diminished parasympathetic heart effects with basal resting tachycardia [349], which, however, has not been documented in patients with SLC5A7-CMS.
Prolyl endopeptidase-like (PREPL) is one of serine peptidases, and its physiological substrate is unknown. The SLC3A1 gene encoding the cystine, dibasic, and neutral amino acid transporter and the PREPL gene are encoded on the opposite strands each other and have an overlap at their 3 ends. Deletion of both genes cause hypotonia-cystinuria syn-drome (HCS) [350]. Deletion of PREPL causes muscle hypotonia [351], whereas deletion of SLC3A1 causes cystinuria [352]. Loss-of-function variants of PREPL do not cause endplate AChR deficiency, but inhibit refilling of ACh to synaptic vesicles, reduce the number of releasable ACh quanta, and decrease the probability of vesicular release [351].

Clinical Features and Therapies
CHAT-CMS has been reported in 19 original articles since 2001 [45, [140][141][142]340,343,[353][354][355][356][357][358][359][360][361][362][363][364]. A follow-up study of 11 patients with CHAT-CMS for maximum of 12 years showed two forms of disease [358]. It can present in neonates with episodic apnea, respiratory distress, swallowing difficulty, and limb muscle weakness. It can also start in infancy with episodic apnea, and mild limb muscle weakness. The milder infantile form may show progressive muscle weakness with wheelchair dependency. Episodic apnea is frequently misdiagnosed as epilepsy [358]. Episodic apnea, however, is observed in other groups of CMS, and is not unique to CHAT-CMS. Similar to SCN4A-CMS, about half of CHAT-CMS patients show no decremental response to low-frequency RNS, and require high-frequency RNS at 10 Hz or more. A respiratory monitor is required for neonatal and infantile episodic apnea. ChEIs and amifampridine are effective in most patients with CHAT-CMS [340,341].
SLC5A7-CMS has been reported in 12 patients in 10 pedigrees since 2016 [41, 346,368,369]. Typical clinical features include neonatal-onset episodic apnea, muscle hypotonia, muscle weakness, and weak crying. Some patients have arthrogryposis and congenital malformations and die in infancy, and some patients show developmental delay. Progressive brain atrophy was reported in a case with SLC5A7-CMS, which was likely due to repeated apneustic attacks [346]. In addition, repeated intestinal perforations were reported in two patients of SLC5A7-CMS in a single pedigree [346]. Among 6 patients with SLC5A7-CMS, in whom RNS results were documented, 5 patients showed decremental CMAP to lowfrequency RNS [41, 346,368], and a single patient showed decremental CMAP only after RNS at 20 Hz for 10 sec [41,365]. ChEIs are effective [41, 346,368], and ephedrine has an additional effect [346]. Amifampridine is ineffective [346].
PREPL-CMS without pathogenic variants SLC3A1 has been reported in 11 patients since 2014 [33,351,[370][371][372][373][374][375][376]. Similarly, PREPL-CMS with SLC3A1 deletion, the diagnosis of which is HCS, has been reported in 7 patients since 2014 [33,351]. PREPL-CMS is characterized by fluctuating muscle weakness and feeding difficulty since birth, and sometimes requires respiratory support. Patients show palpebral ptosis, nasal voice, swallowing difficulty and facial muscle weakness, and sometimes proximal limb muscle weakness. Intelligence is normal or slightly affected. CMAP decreases with low-frequency RNS [371,376]. In a patient with PREPL-CMS, however, low-frequency RNS elicited decremental CMAP only after RNS at 20 Hz for 2 min [376]. Ten patients with PREPL-CMS including HCS were initially diagnosed as Prader-Willi syndrome [33]. ChEIs are effective [35,351,372,374]. The first PREPL-CMS patient could discontinue ChEI at age 12 months, although muscle weakness was still present [351]. Synaptotagmin 2 (SYT2) senses Ca 2+ ions entering into the nerve terminal through P/Q-type calcium channel and triggers the formation of the SNARE complex that releases ACh in synaptic vesicles to the synaptic space. Hemiallelic pathogenic variants were identified in SYT2 in patients with CMS resembling LEMS [56,57]. Functional analysis with Drosophila showed that the variants indeed affected the release of synaptic vesicles [56,57].
The SNARE complex is made of SNAP25, syntaxin, and synaptobrevin (vesicleassociated membrane protein 1, VAMP1). Hemiallelic de novo loss-of-function missense variants of SNAP25 cause CMS with developmental delay and ataxia [15]. SNAP25 has two splicing isoforms: SNAP25A transcript includes 118-bp exon 5A, whereas SNAP25B transcript includes 118-bp exon 5B. Embryonic SNAP25A is switched to adult-type SNAP25B after birth. Pathogenic variants in exon 5B encoding SNAP25B cause CMS [15]. t-SNARE liposome containing a variant SNAP25B failed to properly fuse to v-SNARE liposome induced by calcium ions. In addition, bovine chromaffin cells expressing a variant SNAP5B showed compromised exocytosis in response to depolarization.
Another component of the SNARE complex, syntaxin 1, is bent in the middle and is in a closed conformation at rest. Munc18-1 stabilizes syntaxin 1 in a closed state. In response to the entry of Ca 2+ ions to the nerve terminal, Munc13-1 (UNC13A) binds to syntaxin 1 and displaces Munc18-1, which stabilizes the open conformation of syntaxin 1 [377]. Biallelic truncation variants of UNC13A caused severe muscle weakness at birth, microcephaly, hypoplastic corpus callosum, enhanced excitation of cerebral cortex [11]. A microelectrode study of biopsied skeletal muscle showed that UNC13A-CMS decreased the quantal contents of synaptic vesicles but spared the release probability of synaptic vesicles. In contrast to UNC13A-CMS caused by biallelic truncation variants, hemiallelic pathogenic missense variants of UNC13A do not cause CMS but cause dyskinesia, developmental delay, and autism [378].
Laminin α5 (LAMA5) is highly expressed at the NMJ. Knockout of Lama5 results in embryonic lethality in mice [386], whereas muscle-specific knockout of both Lama4 and Lama5 markedly affect the postsynaptic structure [387]. Muscle-specific knockout of Lama5 alone does not show any motor deficit, but differentiation of the nerve terminal is compromised, and the nerve terminal is juxtaposed to only a part of motor endplate [387]. In LAMA5-CMS, quantal release of ACh is markedly reduced. In addition, although the junctional folds of the motor endplate are spared, the motor endplate is not covered by the nerve terminal or covered by a small nerve terminal [13]. Synaptic vesicle glycoprotein 2A (SV2A) binds to synaptotagmin, and pathogenic variants of LAMA5 impairs binding to SV2A [13].
SNAP25-CMS was reported in a single patient in 2014 [15] and another in 2022 [54]. Both were caused by hemiallelic pathogenic variants. SNAP25-CMS shows severe muscle hypotonia and muscle weakness at birth, and arthrogryposis multiplex. A case in 2014 could walk with a walker at age 7 years, and sometimes had palpebral ptosis [15]. In a case in 2014, low-frequency RNS caused a decremental CMAP, but high-frequency RNS was not performed [15]. In a case in 2022, no RNS studies were performed [54]. ChEI was ineffective, but amifampridine was effective [15]. A case in 2022 died at age 6 days due to respiratory distress [54].
UNC13A-CMS was reported in a single patient in 2016 [11]. UNC13A-CMS showed severe muscle hypotonia and muscle weakness at birth, and microcephaly and hypoplastic corpus callosum. Low-and high-frequency RNS showed decremental and incremental CMAP responses, respectively. EEG showed sharp waves, but no epileptic attacks were noted. ChEI and amifampridine improved decremental CMAP in response to RNS, but minimally improved clinical symptoms. The patient died at age 50 months due to respiratory failure.
RPH3A-CMS was reported in a 11-year-old female [12]. She had limb muscle weakness, nasal voice, and intolerance to exercises since age 3 years. She had learning disabilities. No palpebral ptosis or external ophthalmoplegia was noted. She had mild proximal limb muscle weakness and cervical muscle weakness. Although the association to CMS is unknown, repeated abdominal pain and hyperglycemia are also noted. CMAP amplitudes were not decreased at 2 Hz RNS, but were increased at 30 Hz RNS, as observed in LEMS. Salbutamol (albuterol) was effective, and other drugs were not used.
LAMA5-CMS was reported in a single patient in 2017 [13]. The patient was noted with weak cry and was dependent on a respirator. A brother died of muscle weakness, but details were unknown. Minor facial anomalies were also noted. Low-frequency RNS decreased CMAP by maximum 55%. Low-frequency RNS after maximum muscle contraction for 30 sec increased CMAP to 250%. Coadministration of ChEI and amifampridine was effective. Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is a rate-limiting enzyme to produce uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) that is an essential source for N-and O-glycosylations (Figure 4). On the other hand, dolichyl-phosphate N-acetylglucosamine phosphotransferase 1 (DPAGT1) and UDP-N-acetylglucosaminyltransferase subunit (asparagine-linked glycosylation 14 homolog, ALG14) work on the first two steps of adding GlcNAc to dolichyl phosphate in N-glycosylation. Pathogenic variants of DPAGT1 [388] and ALG2 [389] were reported in congenital disorder of glycosylation Ij (CDG Ij) with infantile spasms, developmental delay, microcephaly, and finger malformations. Muscle hypotonia and muscle weakness are documented in CDGs, and some CDGs may have CMS. Alpha-1,3/1,6-mannosyltransferase (asparagine-linked glycosylation 2 homolog, ALG2) add mannose in N-glycosylation pathway. GDP-mannose pyrophosphorylase B (GMPPB) makes GDP-mannose from mannose-1-phosphate and GTP. GMPPB is essential for N-and O-mannosylations. Expression of pathogenic variants of GFPT1 causes abnormal structures of muscle fibers and NMJ in zebra fish [390]. In C2C12 myotubes, knockdown of Gfpt1 [391] and Alg14 [24] markedly reduced cell surface expression of AChR. Although pathogenic variants of these genes cause AChR deficiency in cultured cells, the detailed mechanisms remain to be elucidated.
Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is a rate-limiting enzyme to produce uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) that is an essential source for N-and O-glycosylations (Figure 4). On the other hand, dolichyl-phosphate Nacetylglucosamine phosphotransferase 1 (DPAGT1) and UDP-Nacetylglucosaminyltransferase subunit (asparagine-linked glycosylation 14 homolog, ALG14) work on the first two steps of adding GlcNAc to dolichyl phosphate in Nglycosylation. Pathogenic variants of DPAGT1 [388] and ALG2 [389] were reported in congenital disorder of glycosylation Ij (CDG Ij) with infantile spasms, developmental delay, microcephaly, and finger malformations. Muscle hypotonia and muscle weakness are documented in CDGs, and some CDGs may have CMS. Alpha-1,3/1,6mannosyltransferase (asparagine-linked glycosylation 2 homolog, ALG2) add mannose in N-glycosylation pathway. GDP-mannose pyrophosphorylase B (GMPPB) makes GDPmannose from mannose-1-phosphate and GTP. GMPPB is essential for N-and Omannosylations. Expression of pathogenic variants of GFPT1 causes abnormal structures of muscle fibers and NMJ in zebra fish [390]. In C2C12 myotubes, knockdown of Gfpt1 [391] and Alg14 [24] markedly reduced cell surface expression of AChR. Although pathogenic variants of these genes cause AChR deficiency in cultured cells, the detailed mechanisms remain to be elucidated.
Deficiency of enzymes in O-mannosylation is observed in congenital muscular dystrophy including Fukuyama-type muscular dystrophy and is called dystroglycanopathy. Pathogenic variants of GMPPB cause muscular dystrophy-dystroglycanopathy (MDDG) type 14 [28] with hypoglycosylation of α-dystroglycan and muscular dystrophy in biopsied muscle [25]. Muscle MRI shows displacement of muscle tissue to fibrous and adipose tissues in paravertebral and proximal skeletal muscles [25,407], as observed in muscular dystrophies. In GMPPB-CMS, serum CK is elevated to 2 to 24 times the upper limit of normal (average 10.7 times) [7,8]. In GFPT1-CMS, serum CK is elevated to about 3 times the upper limit of normal [7,8]. The same variant causes GMPPB-CMS [25] and limb-girdle muscle weakness [408], indicating that myasthenia might not be deeply evaluated.
Pathogenic variants of a succinate transporter (SLC25A1) in mitochondrial inner membrane cause combined D-2-and L-2-hydroxyglutaric aciduria (D2L2AD) [415] and CMS. Pathogenic variants of SLC25A1 are predicted to compromise metabolisms of lipid, sterol synthesis, gluconeogenesis, and glycolysis [416], which somehow leads to the development of CMS. Knockdown of Slc25a1 in zebrafish causes aberration in the axonal elongation of spinal motor neurons and compromise the formation of the NMJ [417]. SLC25A1-CMS is thus predicted to be caused by presynaptic defects.

Clinical Features and Therapies
MYO9A-CMS was reported in 3 patients in 2 pedigrees in 2016 [412]. Patients were initially noted with reduced fetal movement before birth and palpebral ptosis at birth. Patients later developed swallowing difficulty, distal and proximal muscle weakness, episodic apnea, respiratory insufficiency, and external ophthalmoplegia. In two patients in a single pedigree, the presence of nystagmus was documented. All patients had developmental delay. ChEI was effective, and combination of ChEI and amifampridine showed marked effects in a patient. However, in a single patient, combination of amifampridine and fluoxetine induced respiratory crisis.
SLC25A1-CMS has been reported in 19 patients in 10 pedigrees since 2014 [417][418][419][420][421]. Limb myasthenia and palpebral ptosis are shared features. External ocular muscles, bulbar muscles, and respiratory muscles are sometimes affected. Developmental delays were also sometimes noted. ChEIs and amifampridine are generally ineffective but are slight effective in some patients.  [422]. Its C-terminal luminal domain interacts with and activates nucleoplasmic TorsinA, an ATPase for the ATPases associated with diverse cellular activities (AAA+) [423]. Knockout of Tor1aip1 in mouse shows endplate AChR deficiency with markedly increased number of myonuclei at the NMJ. Loss-of-function variants of TOR1AIP1 were previously reported to cause limb-girdle muscular dystrophy or dystonia, with cardiomyopathy or a severe multisystem disorder [424][425][426]. Thus, CMS is a novel phenotype caused by pathogenic variants of TOR1AIP1. It is interesting to note that pathogenic variants of LMNA encoding another nucleolar membrane protein, lamin A, also cause multiple disease phenotypes.

Clinical Features and Therapies
TOR1AIP1-CMS was reported in two adult siblings in 2020 [427] and three adult siblings in 2022 [428]. All patients were noted with mild to moderate muscle weakness and myasthenia in limb muscles and took a slowly progressive or stable course. ChEIs were effective [427,428], and addition of salbutamol (albuterol) had no effect [427]. CHD8 is one of ATP-dependent chromatin-remodeling enzymes but binds to β-catenin and suppresses the transcription of target genes of β-catenin [429][430][431]. CHD8 is accumulated at the NMJ and binds to rapsyn through β-catenin [432]. Thus, either transcriptional suppression of β-catenin-target genes or suppressed interaction between β-catenin and rapsyn is likely to account for CHD8-CMS [432]. In addition, knockout of Ctnnb1 encoding β-catenin (βCAT) that binds to CHD8 impairs AChR clustering and release of ACh from the nerve terminal [433]. In Drosophila, Kis, a homolog of CHD8, promoted presynaptic endocytosis at the NMJ [434]. Similarly, in C. elegans, a loss-of-function of Chd8 caused reduced synaptic vesicle recycling [435]. As sated below, a marked effect of amifampridine and lack of effects of ChEIs and salbutamol (albuterol) are also consistent with the notion that the major defect in CHD8-CMS is at the motor nerve terminal [432].

Clinical Features and Therapies
Monozygotic female twins with CHD8-CMS were reported in 2020 [432]. Patients showed neonatal onset respiratory distress, palpebral ptosis, and limb muscle weakness. At age 14 years, when the patients were reported, they showed frequent falling attacks and myasthenia, as well as rapidly progressive scoliosis. ChEI and salbutamol (albuterol) showed no effect, but amifampridine was markedly effective [432]. Hemiallelic loss-offunction variants of CHD8 are also reported in intellectual developmental disorder with autism and macrocephaly (IDDAM) [436,437]. The authors of CHD8-CMS stated as personal communications that muscle hypotonia and muscle weakness were observed in 4 out of 66 patients with pathogenic variants of CHD8 in IDDAM [432].

Clinical Features and Therapies
Three patients with PURA-CMS showing fluctuating muscle weakness were reported in 2022 [55,442]. One patient showed decremental CAMP, as well as R-CMAP that was much higher than that observed in COLQ-CMS and SCCMS [55]. Another patient showed decremental CAMP followed by incremental CMAP [55]. The third patient showed no decremental CMAP at 3 Hz nerve stimulation but showed non-significant incremental CMAP at 30 Hz stimulation [442]. Two patients were neonates [55,442] and the other was 5 years old [55]. The 5-year-old patient became free of symptoms indicating defective NMJ signal transmission after age 2 years. In a patient, ChEI was ineffective, but salbutamol (albuterol) was effective and unnecessitated non-invasive positive pressure ventilation (NIPPV) because of amelioration of episodic apnea [55]. In another patient, ChEI had markedly ameliorated motor deficits [442].

Conclusions
CMS is a group of heterogenous disorders with highly variable clinical phenotypes that require specific treatment for specific pathomechanisms (Table 1)